Process for producing alpha-1,3-glucan polymer with reduced molecular weight

ABSTRACT

A process for producing poly alpha-1,3-glucan with reduced molecular weight is disclosed. The process comprises contacting water, sucrose, a polar organic solvent, and a glucosyltransferase enzyme in a solution to produce poly alpha-1,3-glucan. This contacting step results in the production of poly alpha-1,3-glucan having a reduced molecular weight compared to the molecular weight of a poly alpha-1,3-glucan made in the absence of the polar organic solvent.

This application claims the benefit of U.S. Provisional Application No. 61/873,851, filed Sep. 5, 2013, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention is in the field of enzymatic processes. Specifically, this invention pertains to a process for producing alpha-1,3-glucan polymer in a solution comprising glucosyl transferase, sucrose, and a polar organic solvent.

Reference to Sequence Listing Submitted Electronically

The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named CL5841 USNP_SequenceListing.txt created on Sep. 3, 2014, and having a size of 259 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII-formatted document is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Cellulose, a polysaccharide consisting of beta-1,4-linked glucose formed by natural processes (Applied Fiber Science, F. Happey, Ed., Chapter 8, E. Atkins, Academic Press, New York, 1979), has become the preeminent fiber for use in manufactured textiles, films and resins.

Cellulose and starch exhibit properties that are determined by the nature of their linkage pattern. Starch or amylose consisting of alpha-1,4 linked glucose are not useful for fiber applications because it is swollen or dissolved by water. Cellulose, on the other hand, has a beta-1,4 linkage which provides the crystalline and hydrophobic qualities making cellulose a good structural material. Thus, cellulose is commonly used for textile applications like cotton fiber.

Cellulosic fibers such as cotton and rayon increasingly present sustainability issues with respect to land use and environmental imprint. This may be a significant factor leading to increased level of research into textiles containing polyester fiber blends with cellulosic materials and more sustainable alternatives for cellulosic-derived materials.

Driven by a desire to find new structural polysaccharides using enzymatic syntheses or genetic engineering of microorganisms or plant hosts, researchers have discovered polysaccharides that are biodegradable, and that can be made economically from renewable resource-based feedstocks. One such polysaccharide is poly alpha-1,3-glucan, a glucan polymer characterized by having alpha-1,3-glycosidic linkages. This polymer has been isolated by contacting an aqueous solution of sucrose with a glucosyltransferase enzyme isolated from Streptococcus salivarius (Simpson et al., Microbiology 141:1451-1460, 1995). Films prepared from poly alpha-1,3-glucan tolerate temperatures up to 150° C. and provide an advantage over polymers obtained from beta-1,4-linked polysaccharides (Ogawa et al., Fiber Differentiation Methods 47:353-362, 1980).

U.S. Pat. No. 7,000,000 disclosed the preparation of a polysaccharide fiber comprising hexose units, wherein at least 50% of the hexose units within the polymer were linked via alpha-1,3-glycosidic linkages using an S. salivarius gtfJ enzyme. This enzyme utilizes sucrose as a substrate in a polymerization reaction producing poly alpha-1,3-glucan and fructose as end-products (Simpson et al., 1995).

The production of poly alpha-1,3-glucan for commercial applications using sucrose and gtf enzymes requires a high yield process that produces minimal amounts of by-product such as leucrose as well as the ability to control the polymer length or molecule weight of the resulting poly alpha-1,3-glucan.

Castillo et al. (Journal of Biotechnology 114:209-217, 2004) disclosed that the inclusion of 2-methyl-2-propanol (tert-butyl alcohol) in a reaction for producing levan resulted in levan having an increased molecular weight profile compared to the molecular weight profile of levan made without using tert-butyl alcohol.

Masanori et al. (Japanese Pat. Appl. Publ. No. P2000-175694A) disclosed that the inclusion of dimethyl sulfoxide in a reaction for producing mutan resulted in the production of mutan with increased molecular weight compared to the molecular weight observed in reactions lacking dimethyl sulfoxide. Thus, increasing the molecular weight of certain polysaccharide polymers is possible under certain reaction conditions.

Alternatively, decreasing the molecular weight of polysaccharide polymers is another means by which to control the molecular weight. Accordingly, processes for producing poly alpha-1,3-glucan having reduced molecular weight are desirable as another approach to producing a polysaccharide polymer of desired molecular weight.

SUMMARY OF THE INVENTION

In one embodiment, the invention concerns a process for producing poly alpha-1,3-glucan comprising contacting water, sucrose, a polar organic solvent, and a glucosyltransferase enzyme in a solution, wherein the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan. The poly alpha-1,3-glucan thus produced has a reduced molecular weight compared to the molecular weight of a poly alpha-1,3-glucan that would be produced in the absence of the polar organic solvent. Optionally, the process in this embodiment further comprises the step of isolating the poly alpha-1,3-glucan produced in the contacting step.

In a second embodiment, the polar organic solvent is aprotic. The aprotic polar organic solvent can be acetonitrile, dimethyl sulfoxide, acetone, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, propylene carbonate, or sulfolane, for example.

In a third embodiment, the polar organic solvent is protic. The protic polar organic solvent can be methanol, methyl formamide, ethanol, isopropanol, 1-propanol, tert-butyl alcohol, or formamide, for example.

In a fourth embodiment, the concentration of the polar organic solvent in the solution is about 2% to about 20% by volume. In a fifth embodiment, the concentration of the polar organic solvent in the solution is about 10% by volume.

In a sixth embodiment, the solution has a temperature between about 5° C. to about 50° C.

In a seventh embodiment, the initial concentration of the sucrose in the solution is about 20 g/L to about 400 g/L.

In an eighth embodiment, the molecular weight of the poly alpha-1,3-glucan produced in the contacting step is reduced by at least about 15%. In a ninth embodiment, the molecular weight of the poly alpha-1,3-glucan produced in the contacting step is reduced by at least about 50%.

In a tenth embodiment, the molecular weight is measured as weight average degree of polymerization (DPw). In an eleventh embodiment, the DPw of the poly alpha-1,3-glucan produced in the contacting step is between about 40 and 800.

In a twelfth embodiment, the glucosyltransferase enzyme is a bacterial glucosyltransferase enzyme.

In a thirteenth embodiment, the glucosyltransferase enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:12, SEQ ID NO:2, or SEQ ID NO:32.

BRIEF DESCRIPTION OF THE SEQUENCES

TABLE 1 Summary of Nucleic Acid and Protein SEQ ID Numbers Nucleic Protein acid SEQ SEQ Description ID NO. ID NO. “gtfJ”, Streptococcus salivarius. DNA codon- 1  2 optimized for expression in E. coli. The first 42 amino (1477 aa) acids of the protein are deleted compared to GENBANK Identification No. 47527; a start methionine is included. “0874 gtf”, Streptococcus sobrinus. DNA codon- 3  4 optimized for expression in E. coli. The first 156 (1435 aa) amino acids of the protein are deleted compared to GENBANK Identification No. 450874; a start methionine is included. “2678 gtf”, Streptococcus salivarius K12. DNA codon- 5  6 optimized for expression in E. coli. The first 188 (1341 aa) amino acids of the protein are deleted compared to GENBANK Identification No. 400182678; a start methionine is included. “2919 gtf”, Streptococcus salivarius PS4. DNA 7  8 codon-optimized for expression in E. coli. The first 92 (1340 aa) amino acids of the protein are deleted compared to GENBANK Identification No. 383282919; a start methionine is included. “5926 gtf”, Streptococcus dentirousetti. DNA codon- 9 10 optimized for expression in E. coli. The first 144 (1323 aa) amino acids of the protein are deleted compared to GENBANK Identification No. 167735926; a start methionine is included. “6855 gtf”, Streptococcus salivarius SK126. DNA 11 12 codon-optimized for expression in E. coli. The first (1341 aa) 178 amino acids of the protein are deleted compared to GENBANK Identification No. 228476855; a start methionine is included. “gtfJ-T1”, Streptococcus salivarius. The first 230 13 14 amino acids and the last 384 amino acids of the protein  (905 aa) are deleted compared to GENBANK Identification No. 47527; a start methionine is included. “5926-T1”, Streptococcus dentirousetti. The first 199 15 16 amino acids and the last 417 amino acids of the protein  (851 aa) are deleted compared to GENBANK Identification No. 167735926; a start methionine is included. “wild type gtfJ”, Streptococcus salivarius. GENBANK 17 18 Identification No. 47527. (1518 aa) “2379 gtf”, Streptococcus salivarius. DNA codon- 19 20 optimized for expression in E. coli. The first 203 (1247 aa) amino acids of the protein are deleted compared to GENBANK Identification No. 662379; a start methionine is included. “1724 gtf”, Streptococcus downei. DNA codon- 21 22 optimized for expression in E. coli. The first 162 (1436 aa) amino acids of the protein are deleted compared to GENBANK Identification No. 121724; a start methionine is included. “0544 gtf”, Streptococcus mutans. DNA codon- 23 24 optimized for expression in E. coli. The first 164 (1313 aa) amino acids of the protein are deleted compared to GENBANK Identification No. 290580544; a start methionine is included. “4297 gtf”, Streptococcus oralis. DNA codon- 25 26 optimized for expression in E. coli. The first 228 (1348 aa) amino acids of the protein are deleted compared to GENBANK Identification No. 7684297; a start methionine is included. “5618 gtf”, Streptococcus sanguinis. DNA codon- 27 28 optimized for expression in E. coli. The first 223 (1348 aa) amino acids of the protein are deleted compared to GENBANK Identification No. 328945618; a start methionine is included. “2765 gtf”, unknown Streptococcus sp. C150. DNA 29 30 codon-optimized for expression in E. coli. The first (1340 aa) 193 amino acids of the protein are deleted compared to GENBANK Identification No. 322372765; a start methionine is included. Streptococcus salivarius gtfJ. The first 177 amino 31 32 acids of the protein are deleted compared to (1342 aa) GENBANK Identification No. 47527; a start methionine is included.

DETAILED DESCRIPTION OF THE INVENTION

The disclosures of all patent and non-patent literature cited herein are incorporated herein by reference in their entirety.

As used herein, the term “invention” or “disclosed invention” is not meant to be limiting, but applies generally to any of the inventions defined in the claims or described herein. These terms are used interchangeably herein.

The terms “poly alpha-1,3-glucan”, “alpha-1,3-glucan polymer” and “glucan polymer” are used interchangeably herein. Poly alpha-1,3-glucan is a polymer comprising glucose monomeric units linked together by glycosidic linkages, wherein at least about 50% of the glycosidic linkages are alpha-1,3-glycosidic linkages. Poly alpha-1,3-glucan is a type of polysaccharide. The structure of poly alpha-1,3-glucan can be illustrated as follows:

The terms “glycosidic linkage” and “glycosidic bond” are used interchangeably herein and refer to the type of covalent bond that joins a carbohydrate (sugar) molecule to another group such as another carbohydrate. The term “alpha-1,3-glycosidic linkage” as used herein refers to the type of covalent bond that joins alpha-D-glucose molecules to each other through carbons 1 and 3 on adjacent alpha-D-glucose rings. This linkage is illustrated in the poly alpha-1,3-glucan structure provided above. Herein, “alpha-D-glucose” will be referred to as “glucose”.

The term “sucrose” herein refers to a non-reducing disaccharide composed of an alpha-D-glucose molecule and a beta-D-fructose molecule linked by an alpha-1,2-glycosidic bond. Sucrose is known commonly as table sugar.

The “molecular weight” of the poly alpha-1,3-glucan herein can be represented by various measures such as grams/mole, Daltons, DPw (“weight average degree of polymerization”) and DPn (“number average degree of polymerization”). Various means are known in the art for calculating these molecular weight measurements.

The terms “glucosyltransferase enzyme that synthesizes poly alpha-1,3-glucan”, “glucosyltransferase enzyme”, “gtf enzyme”, “gtf”, and “glucansucrase” are used interchangeably herein. The activity of a gtf enzyme herein catalyzes the reaction of the substrate sucrose to make the products poly alpha-1,3-glucan and fructose. Other products (byproducts) of a gtf reaction can include glucose (where glucose is hydrolyzed from the glucosyl-gtf enzyme intermediate complex), various soluble oligosaccharides (DP2-DP7), and leucrose (where glucose of the glucosyl-gtf enzyme intermediate complex is linked to fructose). Leucrose is a disaccharide composed of glucose and fructose linked by an alpha-1,5 linkage. Wild type forms of glucosyltransferase enzymes generally contain (in the N-terminal to C-terminal direction) a signal peptide, a variable domain, a catalytic domain, and a glucan-binding domain.

The terms “reaction” and “enzymatic reaction” are used interchangeably herein and refer to a reaction that is catalyzed by a glucosyltransferase enzyme. A “reaction solution” as used herein generally refers to a solution comprising at least one active glucosyltransferase enzyme in a buffer solution comprising sucrose, water, and optionally a polar organic solvent. It is in the reaction solution where the step of contacting water, sucrose, a polar organic solvent, and a glucosyltransferase enzyme is performed. The term “under suitable reaction conditions” as used herein, refers to reaction conditions supporting the conversion of sucrose to poly alpha-1,3-glucan using a glucosyltransferase enzyme.

The terms “polar organic solvent” and “water-miscible organic solvent” are used interchangeably herein. A polar organic solvent can be dissolved in water or an aqueous solution. Thus, a polar organic solvent does not separate out into a different phase when added to water or an aqueous solution. A polar organic solvent contains carbon and at least one heteroatom (i.e., non-carbon or—hydrogen atom) such as oxygen, nitrogen, sulfur, or phosphorous. This contrasts with non-polar organic solvents, which generally comprise only carbon and hydrogen atoms. A polar organic solvent typically has a dielectric constant greater than about 4. Polar organic solvents contain dipoles due to polar bonds. In certain embodiments, the polar organic solvent dissolves in water or an aqueous solution at a temperature between about 5° C. to 50° C.

The term “aprotic polar organic solvent” herein refers to a polar organic solvent that does not have suitably labile hydrogen atoms that can form hydrogen bonds. An aprotic polar organic solvent does not contain hydrogen atoms bonded to an atom with electronegative character; e.g., there are no O—H, N—H, or S—H bonds.

The term “protic polar organic solvent” herein refers to a polar organic solvent that has one or more suitably labile hydrogen atoms that can to form hydrogen bonds. A protic polar organic solvent generally contains hydrogen atoms bonded to an atom with electronegative character; e.g., there are O—H, N—H, and/or S—H bonds.

The terms “percent by volume”, “volume percent”, “vol %” and “v/v %” are used interchangeably herein. The percent by volume of a solute in a solution can be determined using the formula: [(volume of solute)/(volume of solution)]×100%.

The terms “percent by weight”, “weight percentage (wt %)” and “weight-weight percentage (% w/w)” are used interchangeably herein. Percent by weight refers to the percentage of a material on a mass basis as it is comprised in a composition, mixture or solution.

The terms “polynucleotide”, “polynucleotide sequence”, and “nucleic acid sequence” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of DNA or RNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.

The term “gene” as used herein refers to a polynucleotide sequence that expresses a protein, and which may refer to the coding region alone or may include regulatory sequences upstream and/or downstream to the coding region (e.g., 5′ untranslated regions upstream of the transcription start site of the coding region). A gene that is “native” or “endogenous” refers to a gene as found in nature with its own regulatory sequences; this gene is located in its natural location in the genome of an organism. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. A “foreign” or “heterologous” gene refers to a gene that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes. The polynucleotide sequences in certain embodiments disclosed herein are heterologous. A “transgene” is a gene that has been introduced into the genome by a transformation procedure. A “codon-optimized gene” is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.

A native amino acid sequence or polynucleotide sequence is naturally occurring, whereas a non-native amino acid sequence or polynucleotide sequence does not occur in nature.

“Coding sequence” as used herein refers to a DNA sequence that codes for a specific amino acid sequence. “Regulatory sequences” as used herein refer to nucleotide sequences located upstream of the coding sequence's transcription start site, 5′ untranslated regions and 3′ non-coding regions, and which may influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, enhancers, silencers, 5′ untranslated leader sequence, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, stem-loop structures and other elements involved in regulation of gene expression.

The term “recombinant” as used herein refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. The terms “recombinant”, “transgenic”, “transformed”, “engineered” or “modified for exogenous gene expression” are used interchangeably herein.

The term “transformation” as used in certain embodiments refers to the transfer of a nucleic acid molecule into a host organism. The nucleic acid molecule may be a plasmid that replicates autonomously, or it may integrate into the genome of the host organism. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms or “transformants”.

The term “recombinant” or “heterologous” refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.

The terms “sequence identity” or “identity” as used herein with respect to polynucleotide or polypeptide sequences refer to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. Thus, “percentage of sequence identity” or “percent identity” refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity.

The Basic Local Alignment Search Tool (BLAST) algorithm, which is available online at the National Center for Biotechnology Information (NCBI) website, may be used, for example, to measure percent identity between or among two or more of the polynucleotide sequences (BLASTN algorithm) or polypeptide sequences (BLASTP algorithm) disclosed herein. Alternatively, percent identity between sequences may be performed using a Clustal algorithm (e.g., ClustalW or ClustalV). For multiple alignments using a Clustal method of alignment, the default values may correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using a Clustal method may be KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids, these parameters may be KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.

Various polypeptide amino acid sequences and polynucleotide sequences are disclosed herein as features of certain embodiments of the disclosed invention. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed herein may be used in certain embodiments. Alternatively, a variant amino acid sequence or polynucleotide sequence in certain embodiments can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a sequence disclosed herein. The variant amino acid sequence or polynucleotide sequence has the same function/activity of the disclosed sequence, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function/activity of the disclosed sequence.

The term “isolated” as used in certain embodiments refers to any cellular component that has been completely or partially purified from its native source (e.g., an isolated polynucleotide or polypeptide molecule). In some instances, an isolated polynucleotide or polypeptide molecule is part of a greater composition, buffer system or reagent mix. For example, the isolated polynucleotide or polypeptide molecule can be comprised within a cell or organism in a heterologous manner. Another example is an isolated glucosyltransferase enzyme.

Embodiments of the disclosed invention concern a process for producing poly alpha-1,3-glucan that comprises contacting water, sucrose, a polar organic solvent, and a glucosyltransferase enzyme that synthesizes poly alpha-1,3-glucan in solution, wherein a poly alpha-1,3-glucan is produced that has a reduced molecular weight compared to the molecular weight of a poly alpha-1,3-glucan that would be produced in the absence of the polar organic solvent. The poly alpha-1,3-glucan produced by the reaction solution can optionally be isolated. Significantly, this process avoids the complexities of introducing enzyme and/or temperature modifications in trying to reduce the molecular weight of poly alpha-1,3-glucan produced by reactions catalyzed by glucosyltransferase enzyme.

This process can alternatively be characterized as synthesizing poly alpha-1,3 glucan from a reaction solution comprising water, sucrose, a polar organic solvent, and a glucosyltransferase enzyme, whereby poly alpha-1,3-glucan is produced that has a reduced molecular weight compared to poly alpha-1,3-glucan that would be produced if the polar organic solvent is not present in the reaction solution.

One of ordinary skill in the art would appreciate that a reaction solution in which there is no added polar organic solvent (pure aqueous) can be a control reaction with respect to the process of the invention disclosed herein. The control reaction in certain embodiments can have comparable features except for the presence of a polar organic solvent (i.e., the only variable is the presence of a polar organic solvent).

The molecular weight of poly alpha-1,3-glucan made by the process of the invention is reduced by at least about 15% in certain embodiments. In other embodiments, the molecular weight of the poly alpha-1,3-glucan produced by the process of the invention is reduced by at least about 50%. Alternatively, the molecular weight of the poly alpha-1,3-glucan produced in the process can be reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% (or any integer between 5% and 70%). The reduced molecular weight can be expressed in terms of the DPw of the produced poly alpha-1,3-glucan. The process of the invention does not produce poly alpha-1,3-glucan having an increased molecular weight, when expressed in terms of DPw for example.

The molecular weight of the poly alpha-1,3-glucan produced by the instant process can be measured as DPw (weight average degree of polymerization). Alternatively, the molecular weight of the poly alpha-1,3-glucan produced by the instant process can be measured in terms of Daltons, grams/mole, or as DPn (number average degree of polymerization). The molecular weight of poly alpha-1,3-glucan produced by the instant process can be measured in DPw and is between about 40 and 800, for example. The DPw of the poly alpha-1,3-glucan produced in the process can alternatively be between about 100-200, 100-300, 100-400, 100-500, 100-600, 100-700, 100-800, 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800.

The poly alpha-1,3-glucan produced by the process of the instant invention is preferably linear/unbranched. The percentage of glycosidic linkages between the glucose monomer units of the poly alpha-1,3-glucan that are alpha-1,3 is at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In such embodiments, accordingly, the poly alpha-1,3-glucan has less than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or 0% (or any integer between 0% and 50%) of glycosidic linkages that are not alpha-1,3. Examples of such non-alpha-1,3 glycosidic linkages include, but are not limited to, beta linkages (e.g., beta-1,2; beta-1,3; beta-1,4; beta-1,6) and other alpha linkages (e.g., alpha-1,2; alpha-1,4; alpha-1,6).

It is understood that the higher the percentage of alpha-1,3-glycosidic linkages present in the poly alpha-1,3-glucan, the greater the probability that the poly alpha-1,3-glucan is linear, since there are lower occurrences of certain glycosidic linkages forming branch points in the polymer. In certain embodiments, the poly alpha-1,3-glucan has no branch points or less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% branch points as a percent of the glycosidic linkages in the polymer. Examples of branch points include alpha-1,6 branch points, such as those that are present in mutan polymer.

The glycosidic linkage profile of the poly alpha-1,3-glucan can be determined using any method known in the art. For example, the linkage profile can be determined using methods that use nuclear magnetic resonance (NMR) spectroscopy (e.g., ¹³C NMR or ¹H NMR). These and other methods that can be used are disclosed in Food Carbohydrates: Chemistry, Physical Properties, and Applications (S. W. Cui, Ed., Chapter 3, S. W. Cui, Structural Analysis of Polysaccharides, Taylor & Francis Group LLC, Boca Raton, Fla., 2005), which is incorporated herein by reference.

The poly alpha-1,3-glucan produced in the disclosed process can be either soluble or insoluble in most aqueous systems, where insoluble polymer is preferred. In general, the solubility of a glucan polymer in most aqueous systems is related to its linkage type, molecular weight and/or degree of branching. Poly alpha-1,3-glucan is generally insoluble at a DPw of 8 and above in aqueous (or mostly aqueous) solutions at 20° C.

The molecular weight of the poly alpha-1,3-glucan produced by the process of the present invention can be measured using any of several means known in the art. For example, glucan polymer molecular weight can be measured using high-pressure liquid chromatography (HPLC), size exclusion chromatography (SEC), or gel permeation chromatography (GPC).

The yield of the poly alpha-1,3-glucan produced in the disclosed process can be at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, based on the weight of the sucrose used in the process.

The process of the invention comprises contacting a glucosyltransferase enzyme capable of synthesizing poly alpha-1,3-glucan with water, sucrose and a polar organic solvent in a solution. The glucosyltransferase enzyme used herein catalyzes the conversion of sucrose to poly alpha-1,3-glucan. An example of such a glucosyltransferase enzyme is wild type gtfJ expressed by Streptococcus salivarius (GENBANK Identification No. 47527, SEQ ID NO:18; Simpson et al., Microbiology 141:1451-1460, 1995). U.S. Pat. No. 7,000,000 (incorporated herein by reference) discloses using a particular gtfJ derived from S. salivarius to produce a poly alpha-1,3-glucan that is suitable for use as a spinnable fiber.

Any glucosyltransferase enzyme may be used in the process of the invention disclosed herein. Preferably, the enzyme is fungal or bacterial. Such an enzyme may be derived from a Streptococcus species, Leuconostoc species or Lactobacillus species, for example. Examples of Streptococcus species from which the glucosyltransferase may be derived include S. salivarius, S. sobrinus, S. dentirousetti, S. downei, S. mutans, S. oralis and S. sanguinis. Examples of Leuconostoc species from which the glucosyltransferase may be derived include L. mesenteroides, L. amelibiosum, L. argentinum, L. carnosum, L. citreum, L. cremoris, L. dextranicum and L. fructosum. Examples of Lactobacillus species from which the glucosyltransferase may be derived include L. acidophilus, L. delbrueckii, L. helveticus, L. salivarius, L. casei, L. curvatus, L. plantarum, L. sakei, L. brevis, L. buchneri, L. fermentum and L. reuteri.

The glucosyltransferase enzyme used in certain embodiments of the invention comprises, or consists of, the amino acid sequence provided in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32. Alternatively, the glucosyltransferase enzyme comprises, or consists of, an amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32, wherein the glucosyltransferase enzyme has activity. The glucosyltransferase enzyme used in certain embodiments comprises an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:12, SEQ ID NO:2, or SEQ ID NO:32. In still another aspect, a glucosyltransferase enzyme may be any of the above-identified amino acid sequences and further include 1-300 (or any integer there between) residues on the N-terminus and/or the C-terminus and still retain activity. Such additional residues may be from a corresponding wild type sequence from which the glucosyltransferase enzyme is derived, or may be another sequence such as an epitope tag (at either N- or C-terminus) or a heterologous signal peptide (at N-terminus), for example.

In another aspect a glucosyltransferase enzyme could be a variant enzyme, i.e., one that is non-naturally occurring but retains activity. For example, it could be modified through a truncation or a deletion. Such a variant enzyme may lack a number of amino acids at the N- and/or C-terminus (truncation or internal deletion) compared to the wild type sequence from which the variant enzyme was derived. For example, a glucosyltransferase enzyme used herein may lack amino acids in the signal peptide and/or variable domain that are otherwise present in the corresponding wild type form of the enzyme. This is an example of an enzyme with an N-terminal truncation or internal deletion. As another example, a glucosyltransferase enzyme used in the instant process may lack amino acids in the glucan-binding domain. This is an example of an enzyme with a C-terminal truncation or internal deletion. Other examples of enzymes that can be used are those having both N- and C-terminal truncations or deletions. All the various modified glucosyltransferase enzymes disclosed herein, such as the above-described truncated and internally deleted variants, have glucosyltransferase activity.

The glucosyltransferase enzyme in certain embodiments is encoded by the polynucleotide sequence provided in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31. Alternatively, the glucosyltransferase enzyme is encoded by a polynucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31.

The glucosyltransferase enzyme in certain embodiments synthesizes poly alpha-1,3-glucan in which at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any integer between 50% and 100%) of the constituent glycosidic linkages are alpha-1,3 linkages. In such embodiments, accordingly, the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan in which there is less than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of glycosidic linkages that are not alpha-1,3. Examples of such non-alpha-1,3 glycosidic linkages include beta linkages (e.g., beta-1,2; beta-1,3; beta-1,4; beta-1,6) and other alpha linkages (e.g., alpha-1,2; alpha-1,4; alpha-1,6).

In other aspects, the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan with no branch points or less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% branch points as a percent of the glycosidic linkages in the polymer. Examples of branch points include alpha-1,6 branch points, such as those that are present in mutan polymer.

One or more different glucosyltransferase enzymes may be used in the solution in which the enzyme is contacted with water, sucrose and a polar organic solvent. The glucosyltransferase enzyme in certain embodiments does not have, or has very little (less than 1%), dextransucrase, reuteransucrase, or alternansucrase activity. The glucosyltransferase enzyme in certain embodiments does not produce, or produces very few (less than 1% or 2%), glycosidic linkages that are not alpha-1,3 glycosidic linkages. Nor is the glucosyltransferase a wild type enzyme derived from S. mutans in certain embodiments.

The glucosyltransferase enzyme used in the instant process can be primer-independent or primer-dependent. A primer-dependent glucosyltransferase enzyme requires the presence of an initiating molecule in the reaction solution to act as a primer for the enzyme during glucan polymer synthesis. The term “primer” as used herein refers to any molecule that can act as the initiator for a glucosyltransferase enzyme. Primer-independent glucosyltransferase enzymes do not require the presence of a primer to perform glucan synthesis. Primers that can be used in certain embodiments include dextran and other carbohydrate-based primers, such as hydrolyzed glucan.

The glucosyltransferase enzyme used herein may be produced by any means known in the art (e.g., U.S. Pat. No. 7,000,000, which is incorporated herein by reference). For example, the glucosyltransferase enzyme may be produced recombinantly in any bacterial (e.g., E. coli such as TOP10, Bacillus sp.) or eukaryotic (e.g., yeasts such as Pichia sp. and Saccharomyces sp.) heterologous gene expression system. One of the above-listed nucleic acid sequences can be used for this purpose, for example. Alternatively, the glucosyltransferase enzyme may be obtained from a species that naturally produces a glucosyltransferase enzyme.

The glucosyltransferase enzyme used herein may be purified and/or isolated prior to its use, or may be used in the form of a cell lysate, for example. A cell lysate or extract may be prepared from a bacteria (e.g., E. coli) used to heterologously express the enzyme. For example, the bacteria may be subjected to disruption using a French pressure cell (French press). The glucosyltransferase enzyme is soluble in these type of preparations. The lysate or extract may be used at about 0.15-0.3% (v/v) in a reaction solution for producing poly alpha-1,3-glucan from sucrose. In certain embodiments, a bacterial cell lysate is first cleared of insoluble material by means such as centrifugation or filtration.

In certain embodiments, the heterologous gene expression system may be one that is designed for protein secretion. The glucosyltransferase enzyme comprises a signal peptide (signal sequence) in such embodiments. The signal peptide may be either its native signal peptide or a heterologous signal peptide.

The activity of the glucosyltransferase enzyme can be determined using any method known in the art. For example, glucosyltransferase enzyme activity can be determined by measuring the production of reducing sugars (fructose and glucose) in a reaction solution containing sucrose (50 mg/mL), dextran T-10 (1 mg/mL) and potassium phosphate buffer (pH 6.5, 50 mM), where the solution is held at 22-25° C. for 24-30 hours. The reducing sugars can be measured by adding 0.01 mL of the reaction solution to a mixture containing 1 N NaOH and 0.1% triphenyltetrazolium chloride and then monitoring the increase in absorbance at OD_(480 nm) for five minutes.

A polar organic solvent is used in the disclosed process. The polar organic solvent can be aprotic. Examples of aprotic polar organic solvents that can be used include, but are not limited to, acetonitrile, dimethyl sulfoxide, acetone, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, propylene carbonate, and sulfolane. Other non-limiting examples of aprotic polar organic solvents that can be used include hexamethylphosphoramide, dimethylimidazolidinone (1,3-dimethyl-2-imidazolidinone), dioxane, nitromethane, and butanone. In general, ester, ketone and aldehyde solvents having no acidic hydrogen atom are other examples of aprotic polar organic solvents that can be used. Acetonitrile can be used as a preferred polar organic solvent.

The polar organic solvent can be protic. Examples of protic polar organic solvents that can be used include, but are not limited to, methanol, methyl formamide, ethanol, isopropanol, 1-propanol, tert-butyl alcohol, and formamide. Other non-limiting examples of protic polar organic solvents that can be used include n-butanol, ethylene glycol, 2-methoxyethanol, 1-methoxy-2-propanol, glycerol, 1,2-propanediol and 1,3-propanetriol. In general, alcohols are other examples of protic polar organic solvents that can be used.

One or more polar organic solvents, such as any of those listed above, may be used in performing the disclosed process. In certain embodiments, the concentration of the polar organic solvent in the solution is about 2% to about 20% by volume. The concentration of the polar organic solvent in the solution is about 10% by volume in certain embodiments. Alternatively, the concentration of the polar organic solvent in the solution can be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% by volume. Where two or more polar organic solvents are used, their total concentration may be any of the above volume percent measurements.

The temperature of the reaction solution in which the water, sucrose, polar organic solvent and glucosyltransferase enzyme are contacted can be controlled, if desired. In certain embodiments, the solution has a temperature between about 5° C. to about 50° C. The temperature of the solution in certain other embodiments is between about 20° C. to about 40° C. Alternatively, the temperature of the solution may be about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40° C.

The temperature of the reaction solution may be maintained using various means known in the art. For example, the temperature of reaction solution can be maintained by placing the vessel containing the reaction solution in an air or water bath incubator set at the desired temperature.

The initial concentration of the sucrose in the solution can be about 20 g/L to about 400 g/L, for example. Alternatively, the initial concentration of the sucrose can be about 75 g/L to about 175 g/L. Alternatively still, the initial concentration of the sucrose can be about 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, or 160 g/L. The “initial concentration of sucrose” refers to the sucrose concentration in the solution after all the reaction solution components have been added (water, sucrose, gtf enzyme).

Sucrose used in the reaction solution can be highly pure (≧99.5%) or be of any other purity or grade. For example, the sucrose can have a purity of at least 99.0%, or be reagent grade sucrose. The sucrose may be derived from any renewable sugar source such as sugar cane, sugar beets, cassava, sweet sorghum, or corn. The sucrose can be provided in any form such as crystalline form or non-crystalline form (e.g., syrup or cane juice).

The pH of the solution in which the water, sucrose, polar organic solvent and glucosyltransferase are contacted can be between about 4.0 to about 8.0 in certain embodiments. Alternatively, the pH can be about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. In certain embodiments, the pH of a solution containing the water, sucrose and polar organic solvent may be set before adding the glucosyltransferase enzyme. The pH of the reaction solution can be adjusted or controlled by the addition or incorporation of a suitable buffer, including but not limited to: phosphate, tris, citrate, or a combination thereof. The concentration of the buffer can be from 0 mM to about 100 mM, for example. In certain embodiments, the buffer concentration is about 10, 20, or 50 mM. A suitable amount of DTT (e.g., about 1.0 mM) may also be added to the reaction solution in certain embodiments.

Water, sucrose, a polar organic solvent and a glucosyltransferase enzyme are contacted in a reaction solution. It will be understood that, as the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan, the reaction solution becomes a reaction mixture given that insoluble poly alpha-1,3-glucan falls out of solution as indicated by clouding of the reaction. The contacting step of the disclosed process can be performed in any number of ways. For example, the desired amounts of sucrose and polar organic solvent can first be dissolved in water (optionally, other components may also be added at this stage of preparation, such as buffer components), followed by the addition of the glucosyltransferase enzyme. The solution may be kept still, or agitated via stirring or orbital shaking, for example. The reaction can be, and typically is, a cell-free.

The glucosyltransferase enzyme can be added to water or an aqueous solution (e.g., sucrose in water; sucrose and polar organic solvent in water) that does not contain salt or buffer when initially preparing the reaction solution. The pH of such a preparation can then be modified as desired, such as to pH 5-6 for example. The reaction can be carried out to completion without any added buffer, if desired.

Completion of the reaction in certain embodiments can be determined visually (no more accumulation of precipitated poly alpha-1,3-glucan) and/or by measuring the amount of sucrose left in the solution (residual sucrose), where a percent sucrose consumption of over about 90% can indicate reaction completion. Typically, a reaction of the disclosed process will take about 12, 24, 36, 48, 60, 72, 84, or 96 hours to complete, depending on certain parameters such as the amount of sucrose and glucosyltransferase enzyme used in the reaction.

The percent sucrose consumption of a reaction in certain embodiments of the disclosed process is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Alternatively, the percent sucrose consumption may be >90% or >95%.

In certain embodiments of the disclosed process, the polar organic solvent does not significantly reduce the activity of the glucosyltransferase enzyme in terms of the percent sucrose consumed during the reaction. This is the case, for example, in reactions where the percent sucrose consumed at the completion of the reaction is at least 90% or 95%.

The inclusion of a polar organic solvent in the disclosed process results in reducing the molecular weight of the poly alpha-1,3-glucan produced by a glucosyltransferase enzyme. Another way to reduce the molecular weight in certain embodiments is to increase the reaction temperature. In certain embodiments of the disclosed process, the polar organic solvent enhances such temperature-dependent reduction in poly alpha-1,3-glucan molecular weight. Such enhancement can be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% (or any integer between 5% and 40%) more reduction of molecular weight of poly alpha-1,3-glucan produced in the process containing a polar organic solvent compared to that produced in a corresponding or control process lacking the polar organic solvent. Any of the glucosyltransferase enzymes and polar organic solvents disclosed herein may be used in this aspect of the disclosed process. For example, gtfJ (SEQ ID NO:2) and acetonitrile (e.g., 10 vol %) may be used. The increase in temperature can be by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14° C. in certain embodiments (e.g., from 25° C. to 37° C.).

The poly alpha-1,3-glucan produced in the disclosed process may optionally be isolated. For example, insoluble poly alpha-1,3-glucan may be separated by centrifugation or filtration. In doing so, the poly alpha-1,3-glucan is separated from the rest of the reaction solution, which may comprise water, fructose and certain byproducts (e.g., leucrose, soluble oligosaccharides DP2-DP7). This solution may also comprise residual sucrose and glucose monomer.

Poly alpha-1,3 glucan is a potentially low cost polymer which can be enzymatically produced from renewable resources containing sucrose using glucosyltransferase enzymes. It has been shown that this polymer can form ordered liquid crystalline solutions when the polymer is dissolved in a solvent under certain conditions (U.S. Pat. No. 7,000,000). Such solutions can be spun into continuous, high strength, cotton-like fibers. The poly alpha-1,3 glucan produced using the instant process has comparable utilities. In addition, the poly alpha-1,3 glucan produced herein can be derivatized as described in U.S. Pat. Appl. Publ. Nos. 201 4/01 7991 3 and 2014/0187767, which are both incorporated herein by reference.

EXAMPLES

The disclosed invention is further defined in the following Examples. It should be understood that these Examples, while indicating certain preferred aspects of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

Abbreviations

The meaning of some of the abbreviations used herein is as follows: “g” means gram(s), “h” means hour(s), “mL” means milliliter(s), “psi” means pound(s) per square inch, “wt %” means weight percentage, “μm” means micrometer(s), “%” means percent, “° C.” means degrees Celsius, “mg” means milligram(s), “mm” means millimeter(s), “mL/min” means milliliters per minute, “m” means meter(s), “μL” means microliter(s), “mmol” means millimole(s), “min” means minute(s), “mol %” means mole percent, “M” means molar, “mg/g” means milligram per gram, “rpm” means revolutions per minute, “MPa” means megaPascals.

General Methods

All reagents were obtained from Sigma-Aldrich (St. Louis, Mo.) unless stated otherwise. N,N-Dimethylacetamide was obtained from J. T. Baker (Phillipsburg, N.J.). Sucrose was obtained from VWR (Radnor, Pa.).

Preparation of Crude Extracts of Glucosyltransferase (gtf) Enzymes

The Streptococcus salivarius gtfJ enzyme (SEQ ID NO:2) used in Examples 1 and 2 was expressed in E. coli strain DH10B using an isopropyl beta-D-1-thiogalactopyranoside (IPTG)-induced expression system. SEQ ID NO:2 has an N-terminal 42-residue deletion compared to the S. salivarius gtfJ amino acid sequence in GENBANK Identification No. 47527. Briefly, E. coli DH10B cells were transformed to express SEQ ID NO:2 from a DNA sequence (SEQ ID NO:1) codon-optimized to express the gtfJ enzyme in E. coli. This DNA sequence was contained in the expression vector, pJexpress404® (DNA 2.0, Menlo Park Calif.). The transformed cells were inoculated to an initial optical density (OD at 600_(nm)) of 0.025 in LB medium (10 g/L Tryptone; 5 g/L yeast extract, 10 g/L NaCl) and allowed to grow at 37° C. in an incubator while shaking at 250 rpm. The cultures were induced by addition of 1 mM IPTG when they reached an OD₆₀₀ of 0.8-1.0. Induced cultures were left on the shaker and harvested 3 hours post induction.

GtfJ enzyme (SEQ ID NO:2) was harvested by centrifuging cultured cells (25° C., 16,000 rpm) in an Eppendorf® centrifuge, re-suspending the cells in 5.0 mM phosphate buffer (pH 7.0) and cooling to 4° C. on ice. The cells were broken using a bead beater with 0.1-mm silica beads, and then centrifuged at 16,000 rpm at 4° C. to pellet the unbroken cells and cell debris. The crude extract (containing soluble gtfJ enzyme, SEQ ID NO:2) was separated from the pellet and analyzed by Bradford protein assay to determine protein concentration (mg/m L).

The gtf enzymes used in Example 3 were prepared as follows. E. coli TOP10° cells (Invitrogen, Carlsbad Calif.) were transformed with a pJexpress404®-based construct containing a particular gtf-encoding DNA sequence. Each sequence was codon-optimized to express the gtf enzyme in E. coli. Individual E. coli strains expressing a particular gtf enzyme were grown in LB medium with ampicillin (100 μg/mL) at 37° C. with shaking to OD₆₀₀=0.4-0.5, at which time IPTG was added to a final concentration of 0.5 mM. The cultures were incubated for 2-4 hours at 37° C. following IPTG induction. Cells were harvested by centrifugation at 5,000×g for 15 minutes and resuspended (20% w/v) in 50 mM phosphate buffer pH 7.0 supplemented with dithiothreitol (DTT, 1.0 mM). Resuspended cells were passed through a French Pressure Cell (SLM Instruments, Rochester, N.Y.) twice to ensure >95% cell lysis. Lysed cells were centrifuged for 30 minutes at 12,000×g at 4° C. The resulting supernatant was analyzed by the BCA protein assay and SDS-PAGE to confirm expression of the gtf enzyme, and the supernatant was stored at −20° C.

Analysis of Reaction Profiles

Periodic samples from reactions were taken and analyzed using an Agilent® 1260 HPLC equipped with a refractive index detector. An Aminex® HP-87C column (BioRad, Hercules, Calif.) having deionized water at a flow rate of 0.6 mL/min and 85° C. was used to quantitate the level of sucrose, glucose, leucrose and fructose in the reaction mixtures. An Aminex® HP-42A column (BioRad) having deionized water at a flow rate of 0.6 mL/min and 85° C. was used to quantitate soluble oligosaccharide byproducts (DP2-DP7).

Analysis of Glucan Molecular Weight

Insoluble glucan polymer isolated from reaction mixtures was treated with N,N-dimethylacetamide (DMAc) with 5% lithium chloride (LiCI) at 100° C. for 16 hours to form a glucan polymer solution. This solution (100 μL) was then injected into an Alliance™ 2695 HPLC (Waters Corporation, Milford, Mass.) equipped with a differential refractometer detector operating at 50° C. The mobile phase (DMAc containing 0.11 wt % LiCl) passed at a flow rate of 0.5 mL/min through four styrene-divinyl benzene columns in series; specifically, one KD-802, one KD-801, and two linear KD-806M columns (Shodex, Japan). The polymer molecular weight distribution of the glucan polymer sample was determined by comparison of retention time to a broad glucan standard.

Example 1 Glucan Polymerization Reactions Using gtfJ Enzyme and Polar Organic Solvents

This Example describes producing alpha-1,3-glucan in gtf-catalyzed reactions containing polar organic solvent. Specifically, this example shows that including a polar organic solvent (10 vol %) in a gtf reaction solution reduces the molecular weight of alpha-1,3-glucan synthesized by the reaction. The gtf in this Example was the S. salivarius gtfJ enzyme (SEQ ID NO:2).

The desired amount of sucrose was weighed out and diluted up to 90 mL using deionized water. 10 mL of either a polar organic solvent (see Table 2) or water (control) was then added to bring the total volume to 100 mL. The polar organic solvent used was methanol, methyl formamide, ethanol, DMSO, i-propanol, t-butanol, n-propanol, acetone, formamide, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, propylene carbonate, or sulfolane. Each solution was next transferred to a 125-mL glass bottle equipped with a polypropylene cap. Fermasure™ was then added (0.5 mL/L reaction, which is 500 ppm), and the pH was adjusted to 5.5 using 5 wt % aqueous sodium hydroxide or 5 wt % aqueous sulfuric acid. The glucan polymerization reaction was initiated by adding 0.3 vol % of crude gtf enzyme (SEQ ID NO:2) extract prepared as described in the General Methods section. This extract contained about 2.9 mg/mL of protein. Agitation to the reaction solution was provided using an Innova® 42 incubator shaker controlled at 25° C. The reaction solution was periodically monitored by HPLC as described in the General Methods section.

After the reaction was determined to be complete by either complete consumption of sucrose or no change in sucrose concentration between measurements, the reaction slurry was filtered. The insoluble alpha-1,3-glucan polymer was then washed with water (200 mL) and acetone (50 mL) and dried at 105° C. using a heated balance (Mettler Toledo® HG63) until no mass change was observed.

The polymer molecular weight was measured according to the General Methods and is presented as the degree of polymerization based on weight (DPw, also referred to as “weight average degree of polymerization”), which can be calculated as the average polymer molecular weight divided by the monomer molecular weight. The results of the polymerization reactions are provided in Table 2. In all cases, a lower polymer molecular weight was observed for reactions in which a polar organic solvent was included, as compared to the molecular weight of the polymer produced by the pure aqueous reaction.

TABLE 2 Molecular Weight of alpha-1,3-Glucan Polymer Produced in Reaction Solutions Containing 10 Vol % Organic Solvent % Sucrose Final % Initial sucrose consumption Sucrose Reaction Solvent DPw (g/L) after 24 hr consumption time (hr) Water only 714 100 96 96 24 Methanol 594 98 94 94 96 Methyl formamide 519 111 39 40 48 Ethanol 518 98 93 95 96 DMSO 462 98 ND^(a) 95 65 i-propanol 431 98 82 96 43 t-butanol 424 109 95 96 96 n-propanol 374 98 86 95 96 Acetone 358 99 72 96 44 Formamide 329 103 63 97 67 Acetonitrile 321 100 >95   >95 28 N,N-dimethylformamide 313 102 82 95 88 N,N-dimethylacetamide 304 99 71 92 44 Tetrahydrofuran 286 101 72 96 44 Propylene carbonate 260 99 57 90 88 Sulfolane 236 98 75 95 48 ^(a)Not determined.

The data in Table 2 indicate that the inclusion of a polar organic solvent (10 vol %) in the gtf reaction solution reduced the DPw of the alpha-1,3-glucan polymer produced in the reaction. This reduction was as compared to the DPw of the polymer produced in the control reaction in which no polar organic solvent was added (water only). The DPw reductions observed ranged from about 17% (when methanol was used) to about 67% (when sulfolane was used). In almost every case (except where the added polar organic solvent was methyl formamide), over 90% of the sucrose supplied in each reaction was consumed, indicating that the added polar organic solvent generally did not inhibit complete consumption of sucrose. Furthermore, since many of the solvent systems had sucrose conversion after 24 hours similar to the sucrose conversion of the pure aqueous system (Table 2), but lower polymer DPw, differences in polymer DPw cannot necessarily be attributed to enzyme activity.

Table 2 also indicates that both protic and aprotic polar organic solvents were useful in gtf reaction solutions to reduce the DPw of the alpha-1,3-glucan polymer produced in the reaction. The protic polar organic solvents were methanol, methyl formamide, ethanol, i-propanol (isopropanol), n-propanol (1-propanol), t-butanol (tert-butyl alcohol), and formamide, whereas the aprotic polar organic solvents were DMSO (dimethyl sulfoxide), acetone, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, propylene carbonate, and sulfolane. In general, aprotic polar organic solvents were more effective at reducing polymer DPw when added to the reaction compared to protic polar organic solvents (Table 2). Most of the gtf reactions to which an aprotic polar organic solvent was added produced glucan polymer with a DPw that was reduced by more than 50% compared to the glucan polymer produced in the completely aqueous gtf reaction.

Thus, the addition of a polar organic solvent to a gtf reaction solution reduces the molecular weight of the alpha-1,3-glucan polymer product of the reaction.

Example 2 Glucan Polymerization Reactions Using qtfJ Enzyme and Acetonitrile

This Example describes producing alpha-1,3-glucan in gtf-catalyzed reactions containing the aprotic polar organic solvent, acetonitrile. Specifically, this example shows that increasing levels of acetonitrile lead to greater reductions in polymer molecular weight, and that acetonitrile can reduce the molecular weight of polymer produced in reactions at 37° C.

The procedure described in Example 1 was followed to prepare S. salivarius gtfJ (SEQ ID NO:2) reaction solutions containing acetonitrile. However, the amount of acetonitrile added to the reaction solution was varied from 2-20 mL (for 2, 10, or 20 vol % acetonitrile); the amount of water was adjusted accordingly to maintain a total reaction volume of 100 mL. The results of these reactions are provided in Table 3.

TABLE 3 Molecular Weight of alpha-1,3-Glucan Polymer Produced in Reaction Solutions Containing Various Amounts of Acetonitrile Initial Temperature Volume % sucrose % Sucrose (° C.) Acetonitrile DPw (g/L) consumption 25 0 714 106 96 25 2 702 100 96 25 10 321 100 >95 25 20 186 101 63

As indicated in Table 3, increasing the amount of acetonitrile in the gtf reaction resulted in greater reductions in the alpha-1,3-glucan polymer molecular weight. These results indicate that glucan polymer molecular weight can be controlled by varying the concentration of the added polar organic solvent.

Next, the procedure described in Example 1 was followed to prepare gtf reaction solutions containing acetonitrile, with the exception that the temperature of the reaction was 37° C. (as opposed to 25° C.). The results of these reactions are provided in Table 4.

TABLE 4 Molecular Weight of alpha-1,3-Glucan Polymer Produced at 37° C. in Reaction Solutions Containing Acetonitrile Initial Temperature Volume % sucrose % Sucrose (° C.) Acetonitrile DPw (g/L) consumption 37 0 492 96 97 37 10 187 98 94

As indicated in Table 4, performing the glucan polymerization reaction with acetonitrile at 37° C. led to the production of polymer with reduced molecular weight. As can be seen by comparing the results in Tables 3 and 4 for gtf reaction solutions containing 10 vol % acetonitrile, raising the reaction temperature from 25° C. to 37° C. led to enhanced reduction of the molecular weight of the alpha-1,3-glucan product (from a DPw of 321 at 25° C. to a DPw of 187 at 37° C.). The percent sucrose consumption was over 90% in both the 25° C. and 37° C. reactions.

Table 4 indicates that gtf reaction solutions that were completely aqueous (no polar organic solvent added) also produced polymer with reduced molecular weight, when compared to the same reaction performed at 25° C. Specifically, the DPw of 492 obtained in the 37° C. reaction was lower than the DPw of 714 obtained in the 25° C. reaction (Table 3); this represents a reduction in polymer molecular weight by about 31%. In contrast, the reduction in polymer molecular weight observed between the 25° C. and 37° C. reactions containing 10 vol % acetonitrile was about 42%. The inclusion of a polar organic solvent therefore can enhance the reduction in polymer molecular weight observed when increasing reaction temperature.

Example 3 Glucan Polymerization Reactions Using Various gtf Enzymes

This Example describes producing alpha-1,3-glucan in reactions containing a polar organic solvent and different gtf enzymes. Specifically, this example shows that, in addition to SEQ ID NO:2, other types of gtf enzymes can be used in reactions containing acetonitrile to produce glucan polymer with reduced molecular weight.

The gtf enzymes used in this example were as follows:

The S. salivarius gtfJ enzyme was SEQ ID NO:2, encoded by SEQ ID NO:1.

An N-terminally truncated version of a Streptococcus sobrinus gtf enzyme identified in GENBANK under GI number 450874 was used (SEQ ID NO:4, encoded by SEQ ID NO:3; herein referred to as “0874”).

An N-terminally truncated version of Streptococcus salivarius K12 gtf enzyme identified in GENBANK as a dextransucrase under GI number 400182678 was used (SEQ ID NO:6, encoded by SEQ ID NO:5; herein referred to as “2678”).

An N-terminally truncated version of Streptococcus salivarius PS4 gtf enzyme identified in GENBANK as a putative glucosyltransferase under GI number 383282919 was used (SEQ ID NO:8, encoded by SEQ ID NO:7; herein referred to as “2919”).

An N-terminally truncated version of Streptococcus dentirousetti gtf enzyme identified in GENBANK under GI number 167735926 was used (SEQ ID NO:10, encoded by SEQ ID NO:9; herein referred to as “5926”).

An N-terminally truncated version of Streptococcus salivarius SK126 gtf enzyme identified in GENBANK under GI number 228476855 was used (SEQ ID NO:12, encoded by SEQ ID NO:11; herein referred to as “6855”).

Another version of the S. salivarius gtfJ enzyme used in this study was SEQ ID NO:14 (herein referred to as “gtfJ-T1”). SEQ ID NO:14, compared to the amino acid sequence identified in GENBANK under GI number 47527, is truncated by 230 amino acids at the N-terminus and 384 amino acids at the C-terminus. As with the other gtf enzymes disclosed herein, SEQ ID NO:14 was produced using a DNA that was codon-optimized for expression in E. coli. SEQ ID NO:13 (Table 1), which is representative of a sequence encoding SEQ ID NO:14, was not used for enzyme expression since it is not codon-optimized.

Another version of the S. dentirousetti gtf enzyme used in this study was SEQ ID NO:16 (herein referred to as “5926-T1”). SEQ ID NO:16, compared to the amino acid sequence identified in GENBANK under GI number 167735926, is truncated by 199 amino acids at the N-terminus and 417 amino acids at the C-terminus. As with the other gtf enzymes disclosed herein, SEQ ID NO:16 was produced using a DNA that was codon-optimized for expression in E. coli. SEQ ID NO:15 (Table 1), which is representative of a sequence encoding SEQ ID NO:16, was not used for enzyme expression since it is not codon-optimized.

The procedure described in Example 1 was followed to prepare reaction solutions containing a particular gtf (see Table 5) and acetonitrile (10 vol %). The reactions were performed at 25° C. and the alpha-1,3-glucan produced in each reaction was analyzed for DPw. The results are provided in Table 5.

TABLE 5 Molecular Weight of alpha-1,3-Glucan Polymer Produced in Reaction Solutions Containing Various gtf Enzymes Initial % Sucrose gtf sucrose con- Enzyme SEQ ID NO Solvent^(a) DPw (g/L) sumption 0874 SEQ ID NO: 4 Acetonitrile 52 149 97 None 56 143 90 2678 SEQ ID NO: 6 Acetonitrile 283 149 65 None 657 151 93 2919 SEQ ID NO: 8 Acetonitrile 188 149 94 None 414 152 91 5926 SEQ ID NO: 10 Acetonitrile 57 149 74 None 68 149 96 5926-T1 SEQ ID NO: 16 Acetonitrile 70 149 97 None 108 150 100 6855 SEQ ID NO: 12 Acetonitrile 247 149 96 None 571 151 96 gtfJ SEQ ID NO: 2 Acetonitrile 305 150 71 None 577 151 96 gtfJ-T1 SEQ ID NO: 14 Acetonitrile 252 149 96 None 495 142 94 ^(a)Solvent was completely aqueous (None) or contained 10 vol % acetonitrile (Acetonitrile).

As indicated in Table 5, all the different gtf enzymes produced alpha-1,3-glucan polymer having a reduced molecular weight when used in a reaction solution containing a polar organic solvent (10 vol % acetonitrile), compared to when the enzymes were used in purely aqueous control reactions. All but one of the gtf reactions yielded glucan polymer with a DPw that was reduced by at least 15%.

Most of the reactions containing acetonitrile consumed over 90% of the sucrose supplied in the reaction (Table 5), indicating that the added acetonitrile generally did not inhibit gtf enzyme activity. In certain reactions (gtfs 0874, 2919, 6855, gtfJ-T1), the addition of acetonitrile resulted in sucrose consumption that was equal to or greater than the sucrose consumption that occurred in the purely aqueous reaction.

Table 5 also indicates that the addition of acetonitrile in a gtf reaction reduced the molecular weight of the alpha-1,3-glucan polymer produced regardless of the polymer size generally produced by the gtf. For example, even though gtf enzymes 0874 (SEQ ID NO:4) and 6855 (SEQ ID NO:12) produced glucan polymers of 56 and 571 DPw, respectively, in purely aqueous reaction conditions, both enzymes produced polymer with reduced DPw in reactions containing acetonitrile.

Table 5 also indicates that the addition of acetonitrile in a gtf reaction reduced the molecular weight of the alpha-1,3-glucan polymer produced regardless of the size of the gtf. Specifically, both enzymes gtfJ (SEQ ID NO:2) and its shortened counterpart gtfJ-T1 (SEQ ID NO:14) produced glucan polymer with reduced DPw when acetonitrile was included in the reaction. This was similarly the case with gtf enzymes 5926 (SEQ ID NO:10) and 5926-T1 (SEQ ID NO:16).

Thus, various types of gtf enzymes can be used in reactions containing a polar organic solvent to produce alpha-1,3-glucan polymer with reduced molecular weight. Given that all of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14 and 16 produced alpha-1,3-glucan polymer with reduced molecular weight in reaction solutions containing a polar organic solvent, other gtf enzymes such as SEQ ID NOs:18, 20, 22, 24, 26, 28, 30 and 32 could be used in a similar manner to produce alpha-1,3-glucan polymer with reduced molecular weight. 

What is claimed is:
 1. A process for producing poly alpha-1,3-glucan comprising: (a) contacting water, sucrose, a polar organic solvent, and a bacterial glucosyltransferase enzyme in a solution, wherein the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan, wherein a poly alpha-1,3-glucan is produced having a reduced molecular weight compared to the molecular weight of the poly alpha-1,3-glucan produced in the absence of the polar organic solvent, wherein the glucosyltransferase enzyme comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 12, SEQ ID NO: 2, SEQ ID NO: 32, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO:30; and (b) optionally, isolating the poly alpha-1,3-glucan produced in step (a); wherein the polar organic solvent is selected from the group consisting of acetonitrile, dimethyl sulfoxide, acetone, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, propylene carbonate, sulfolane, hexamethylphosphoramide, 1,3-dimethyl-2-imidazolidinone, nitromethane, butanone, methanol, methyl formamide, ethanol, isopropanol, 1-propanol, tert-butyl alcohol, formamide, n-butanol, ethylene glycol, 2-methoxyethanol, 1-methoxy-2-propanol, glycerol, 1,2-propanediol, and 1,3-propanetriol.
 2. The process of claim 1, wherein the polar organic solvent is selected from the group consisting of acetonitrile, dimethyl sulfoxide, acetone, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, propylene carbonate, sulfolane, methanol, methyl formamide, ethanol, isopropanol, 1-propanol, tert-butyl alcohol, and formamide.
 3. The process of claim 1, wherein the concentration of the polar organic solvent in the solution is about 2% to about 20% by volume.
 4. The process of claim 3, wherein the concentration of the polar organic solvent in the solution is about 10% by volume.
 5. The process of claim 1, wherein the solution has a temperature between about 5° C. to about 50° C.
 6. The process of claim 1, wherein the initial concentration of the sucrose in the solution is about 20 g/L to about 400 g/L.
 7. The process of claim 1, wherein the molecular weight of the poly alpha-1,3-glucan produced in step (a) is reduced by at least about 15%.
 8. The process of claim 1, wherein the molecular weight of the poly alpha-1,3-glucan produced in step (a) is reduced by at least about 50%.
 9. The process of claim 1, wherein the molecular weight is measured as weight average degree of polymerization (DPw).
 10. The process of claim 9, wherein the DPw of the poly alpha-1,3-glucan produced in step (a) is between about 40 and
 800. 11. The process of claim 1, wherein the glucosyltransferase enzyme comprises an amino acid sequence that is at least 98% identical to SEQ ID NO:12, SEQ ID NO:2, SEQ ID NO:32, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30.
 12. The process of claim 2, wherein the glucosyltransferase enzyme comprises an amino acid sequence that is at least 98% identical to SEQ ID NO:12, SEQ ID NO:2, SEQ ID NO:32, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30.
 13. The process of claim 1, wherein the glucosyltransferase enzyme comprises SEQ ID NO:12, SEQ ID NO:2, SEQ ID NO:32, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30.
 14. The process of claim 2, wherein the glucosyltransferase enzyme comprises SEQ ID NO:12, SEQ ID NO:2, SEQ ID NO:32, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30. 